Mono modified exendin with polyethylene glycol or its derivatives and uses thereof

ABSTRACT

Disclosed herein are exendin singly modified with polyethylene glycole or a derivative thereof, a method for the preparation of the same, and uses thereof. Exendin modified at lysine (27) with polyethylene glycol shows biological activity similar to that of natural exendin, but is improved in half life. In addition, the modification position and the number of PEG or its derivative are restricted so as to minimize the side effects caused by a variety of combinations of such factors. The exendin is useful in the prevention and treatment of diseases caused by the over-secretion of insulin, or diseases caused due to a decrease in plasma glucose level, the inhibition of gastric or intestinal motility, the promotion of satiety, or the inhibition of food intake, especially diabetes, obesity and irritable colon syndrome.

TECHNICAL FIELD

The present invention relates to an exendin which is modified singlywith polyethylene glycol or a polyethylene glycol derivative, a methodfor the preparation of the modified exendin, and the use of the modifiedexendin.

BACKGROUND ART

Glucagon-like peptide-1 (hereinafter referred to as GLP-1) functions toinduce various biological effects, including the stimulation of insulinsecretion, the suppression of glucagon secretion, the promotion ofsatiety, the inhibition of gastric or intestinal motility, theaugmentation of glucose use, and the induction of weight loss. Also,GLP-1 is known to have functions of preventing the degeneration ofpancreatic cells caused by the progression of type II diabetes, that is,non-insulin dependent diabetes mellitus (NIDDM), and of promoting thegrowth of nascent-cells so as to recover insulin secretion.Particularly, the conspicuous feature of GLP-1 resides in the ability tostimulate insulin secretion without the concomitant induction ofhypoglycemia, which is a major risk of insulin therapy or oral therapyfor inducing an increase in insulin expression. In addition, none of theadverse events associated with the long-term administration of thehypoglycemic agent sulfonylurea, such as the apoptosis and necrosis ofpancreatic β-cells, are found with GLP-1. Therefore, GLP-1 is regardedas very useful in the treatment of type II diabetes.

However, therapy with GLP-1 is restricted in utility not only becausethe activity of GLP-1 itself is insufficient, but also because twotruncated, native GLP-1 molecules, GLP-1 (7-37)OH and GLP-1(7-36)NH₂,have very short plasma half lives. In detail, GLP-1 is one of thesubstrates of endogenous dipeptidyl peptidase-IV, being inactivated bythe removal of the N-terminal histidine-alanine dipeptide moiety (aa 7and 8), which is known to be a major cause of the short biological lifespan thereof (O'Harte et al., 2000).

There are many approaches to reduce the degradation of GLP-1 or toextend the plasma lifespan of GLP-1 while maintaining the biologicalactivity thereof, including the use of DPP-IV inhibitors (P93/01,NVP-LAF237, NVP-DPP728, 815541A, 823093, MK-0431, etc.), and the use ofligands acting to GLP-1 receptors or GLP-1 derivatives (exendin,liraglutide, GLP-1/CJC-1131, etc.).

Exendins, first discovered by John Eng (U.S. Pat. No. 5,424,286), are afamily of polypeptides capable of reducing the blood level of glucose.Exendin-4 has the following amino acid sequence, sharing partialhomology (53%) with GLP-1(7-36)NH₂ (Goke et al., 1993).

His1-Gly-Glu-Gly-The-Phe-The-Ser-Asp-Leu-Ser-Lys12-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys27-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂

Exendins are found in the venom of the beaded lizard and the gilamonster: exendin-3 is extracted from the poison of the beaded lizard,Heloderma horridum and exendin-4 is present in the gila monster,Heloderma suspectum. Exendin-4 is different from exendin-3 only in aminoacid position 2 and 3. In mammals, exendins are resistant to DPP-IVdigestion so that their half life is longer than that of GLP-1, which is2 min or less (Kieffer T J et al., 1995). In an in vivo test, exendinswere found to have a half life of 2˜4 hrs and reach a sufficient levelin the blood upon two or three abdominal administrations per day(Fineman M S et al., 2003). In addition, it is known that exendin-4functions to regulate gastrointestinal motility, decrease food intakeand inhibit blood glucagon (U.S. Pat. Nos. 6,858,576, 6,956,026 and6,872,700). With regard to the effect of exendin-4 on glycemic control,HbA_(1c) levels, the amounts of hemoglobin bound to glucose in blood,were measured to decrease by 1% or less in both groups administered withexendin-4 alone and in combination with an anti-diabetic agent, such assulfonylurea or metformin (Egan J M et al., 2003). Recently, the sale ofsynthetic exendin-4 under the brand name of Byetta has been approved bythe FDA.

Polyethylene glycol (PEG), a polymer having the chemical structure ofHO—(—CH₂CH₂O—)_(n)—H, is strongly hydrophilic, and thus it can increasethe solubility of medicinal peptides when it is coupled therewith. PEG,when properly coupled with medicinal peptides, increases the molecularweight of the modified peptides to protect them from renal filtration,cells recognizing exogenous antigens, antibodies, and enzymaticdegradation while their major biological functions, such as enzymaticactivity and receptor binding are maintained. When its molecular weightfalls within the range from 1,000 to 100,000, PEG can be properlycoupled with peptides. PEG with a molecular weight of 1,000 or higher isknown to have very low toxicity. PEG ranging in molecular weight from1,000 to 6,000 is distributed throughout the body and metabolized in thekidneys. Particularly, PEG having about MW 40,000 is distributed inblood and the liver and the metabolism thereof is conducted in theliver.

Generally, when administered via parenteral routes, medicinally orpharmaceutically useful proteins may be antigenic in the body, for themost part, poor in water solubility and have a short retention time inthe body. Approaches to overcome these shortcomings are under study.U.S. Pat. No. 4,179,337 teaches that when proteins or enzymes conjugatedwith PEG are used as therapeutics, they enjoy advantages from PEG,including a decrease in antigenicity and an increase in water solubilityand in retention time. Since this patent was granted, many attempts havebeen made to couple biologically active proteins with polyethyleneglycolto overcome various shortcomings. For instance, ribonuclease andsuperoxide dismutase were coupled with PEG (Veronese et al., 1985) andwhen coupled with polymers including PEG, proteins were reported to showincreased water solubility in U.S. Pat. Nos. 4,766,106 and 4,917,888. Inaddition, it was disclosed that recombinant proteins coupled with PEG orother polymers are decreased in antigenicity and increased in retentiontime in U.S. Pat. No. 4,902,502.

Despite such advantages, PEG limits the function of the proteinsconjugated therewith. In detail, PEG is conjugated with a protein via acovalent bond to a lysine residue(s) of the protein. When the lysineresidue is directly responsible for the activity of the protein, theprotein conjugated with PEG cannot perform the biological function anymore. Furthermore, because the bonding of PEG with lysine residues takesplace randomly, many kinds of PEG-protein conjugates may result, makingit complicated to separate the desired ones from the conjugate mixture.

There have been many attempts to conjugate GLP-1 or analogues thereofand exendin, which are therapeutically useful and have a short half lifewith PEG. PCT Publication No. 04/093823 reports that, when coupled withone or more polyethyleneglycol molecules, GLP-1 compounds or derivativesthereof were increased in half life and underwent degradation at lowrates. The incorporation of one or two cysteine residues into specificamino acid residues of a peptide of interest provides one or two thiolgroups, which then act as sites with which PEG or derivatives thereof(particularly, PEG-maleimide) can form a covalent bond so as to producepolyethylene-glycolated GLP-1 compounds. In an alternative, GLP-1,analogues or fragments thereof are covalently bonded to polyethyleneglycol or polyethylene glycol derivatives at lysine residues or thecarboxyl terminus to produce modified molecules which are extended inhalf life and decreased in degradation rate. In PCT Publication No.04/093823, it was disclosed that PEG or derivatives thereof. Can formcovalent bonds with cysteine resides at positions 26 and 34, lysineresides at positions 18, 22 and 26, or the carboxyl terminal residue ofGLP-1 or analogues. It is also suggested that one peptide molecule maybe conjugated with one to six PEG molecules, which preferably range inmolecular weight from 20,000 to 40,000 daltons. Thepolyethylene-glycolated GLP-1 compounds prepared according to the methodof PCT Publication No. 04/093823 were disclosed to have longer halflives and lower degradation rates in relation to natural GLP-1 orVal₈-GLP-1(7-37)OH, while retaining all or some of the biologicalactivity of natural GLP-1.

U.S. Pat. No. 6,924,264 addresses novel modified exendin and exendinagonist analogs. The modified exendin-4 has three amino acid residues(N-terminal histidine, lysine 12 and 27) capable of being linked to PEG.The PEG used in U.S. Pat. No. 6,924,264 has a molecular weight rangingfrom 5000 to 12,000 daltons and can form covalent bonds withepsilon-amino groups of two lysine residues. In contrast to naturalGLP-1, which undergoes primary proteolysis, the exendins increased inmolecular weight are removed from plasma through renal filtration.

As described above, various approaches can produce exendins which areincreased in lifespan and improved in efficiency in comparison withnatural exendins. However, when two or more PEG molecules are covalentlybonded to biologically active pepti des, such as GLP-1 or exendins, theresulting conjugates may show disadvantageous properties insufficient tobe used as drug for example, may be decreased in stability andbiological activity.

Leading to the present invention, the intensive and thorough researchinto an improvement in the pharmaceutical effect of exendin, conductedby the present inventors, resulted in the finding that the modificationof exendin at a specific amino acid position with PEG or a derivativethereof produces PEG-exendin conjugates which are improved in retentiontime within the body without loss of the biological activity of naturalexendin, and have exellent pharmacokinetic profiles and pharmaceuticaleffects, thereby decreasing in dose or administration number incomparison with natural exendin.

DISCLOSURE OF INVENTION Technical Problem

It is an object of the present invention to provide exendin singlymodified with polyethylene glycol or a derivative thereof.

It is another object of the present invention to provide a method forthe preparation of the exendin singly modified with polyethylene glycolor a derivative thereof.

It is a further object of the present invention to provide uses of theexendin singly modified with polyethylene glycol or a derivativethereof.

Technical Solution

In accordance with an aspect thereof, the present invention providesexendin singly modified with polyethylene glycol or a derivativethereof.

In accordance with another aspect thereof, the present inventionprovides a method for preparing exendin singly modified withpolyethylene glycol or a derivative thereof.

In accordance with a further aspect thereof, the present inventionprovides uses of exendin singly modified with polyethylene glycol or aderivative thereof.

ADVANTAGEOUS EFFECTS

Exendin modified at lysine 27 with polyethylene glycol shows biologicalactivity similar to that of natural exendin, but is improved in halflife. Also, the modification position and the number of PEG or itsderivative are restricted so as to minimize the side effects caused by avariety of combinations of such factors. The exendin is useful in theprevention and treatment of diseases caused by the over-secretion ofinsulin, or diseases caused due to a decrease in plasma glucose level,the inhibition of gastric or intestinal motility, the promotion ofsatiety, or the inhibition of food intake, especially diabetes, obesityand irritable colon syndrome.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 is an HPLC chromatogram of the exendin-4 modified at theN-terminus with PEG and a reaction mixture after the N-terminalreaction;

FIG. 2 is an HPLC chromatogram of the products resulting from thereaction of PEG with lysine residues of exendin-4.

FIG. 3 shows MALDI-TOF mass spactra of the exendin-4 conjugates digestedwith lysine-C;

FIG. 4 shows MALDI-TOF spectra of the exendin-4 modified singly atlysine-27 with PEG, prepared in Example 2;

FIG. 5 shows insulin secretory capacities of natural exendin-4 andPEG-modified exendin-4;

FIG. 6 shows binding behaviors of natural exendin-4 and PEG-modifiedexendin-4 to the GLP-1 receptor located on insulin secreting cellsurfaces;

FIG. 7 shows glucose tolerance measured in animal models administeredwith the exendin-4 modified singly at different positions with PEG;

FIG. 8 shows areas under the glucose curve plotted from 0 to 180 min onthe basis of the result of FIG. 7;

FIG. 9 shows the results of an assay of PEG-modified exendin-4 forinsulin secretory capacity according to the molecular weight of PEG;

FIG. 10 shows the results of an assay of PEG-modified exendin-4 forbinding to GLP-1 receptor according to the molecular weight of PEG;

FIG. 11 shows changes in the blood glucose level of test animalsadministered with exendin-4 modified with PEG having different molecularweights;

FIG. 12 shows changes in the blood glucose level of test animalssubcutaneously administered with natural exendin-4, PEG2k-exendin-4 andPEG20k-exendin-4;

FIG. 13 shows the binding affinity of exendin-4 modified with twodifferent PEG molecules, each having a mean molecular weight of 20000daltons, for the receptor of insulin secretory cells;

FIG. 14 shows anti-diabetic activity of exendin-4 singly modified withPEG of different molecular structures in test animals;

FIG. 15 shows pharmacokinetic behaviors of the exendin-4 singly modifiedwith 20 kDa PEG of different molecular structures;

FIG. 16 shows the dose-dependent insulin secretory capacity of naturalexendin-4 and the exendin-4 modified with 23 kDa tri-branched PEG;

FIG. 17 shows measurements of anti-diabetic activity in animalsadministered with exendin-4 modified at lysine-27 with tri-branched PEGhaving various molecular weights; and

FIG. 18 shows measurements of anti-diabetic activity in animalsadministered with various doses of exendin-4 modified at lysine-27 withtri-branched PEG.

BEST MODE FOR CARRYING OUT THE INVENTION

In accordance with an aspect thereof, the present invention is directedto exendins modified singly with PEG or PEG derivatives, orpharmaceutically acceptable salts thereof.

In an embodiment of this aspect, the modified exendins may be derivedfrom natural or recombinant exendins. In any case, the exendins arepreferably based on exendin-4.

In another embodiment, exendin-4 is preferably modified at position 27with PEG or a PEG derivative. Exendin-4 has three sites which can bemodified with PEG or PEG derivatives (N-terminal histidine, and lysineresidues at positions 12 and 27). However, if modified at two or moresites with PEG or its derivatives, exendin-4 is decreased in stabilityand biological activity for the target molecule to such a degree that itprevents the resulting molecules from being applied to pharmaceuticaluses.

In a further embodiment of the present invention, the PEG which canmodify exendins may be a straight or a branched type. In the lattercase, PEG may be structured to have two or more branches. Preferable isPEG having three branches.

In addition, the PEG or derivatives thereof in accordance with anembodiment of the present invention preferably range in molecular weightfrom 2 to 60 kDa, and more preferably from 20 to 45 kDa. If themolecular weight is below the lower limit, the modified exendin is notgreatly increased in half life compared with natural exendin. On theother hand, when the molecular weight exceeds the upper limit, themodified exendin may show low stability and biological activity withregard to target molecules.

Examples of the polyethylene glycol derivatives used in the presentinvention include methoxypolyethylene glycol succinimidylpropionate,methoxypolyethylene glycol N-hydroxysuccinimide, methoxypolyethyleneglycol propionaldehyde, methoxypolyethylene glycol maleimide, andmulti-branched types thereof, but are not limited thereto. Preferableare methoxypolyethylene glycol succinimidylpropionate,methoxypolyethylene glycol N-hydroxysuccinimide, methoxypolyethyleneglycol pro-pionaldehyde, bi-branched methoxypolyethylene glycolN-hydroxysuccinimide or tri-branched polyethyleneglycol represented bythe following chemical formula 1.

(wherein

n is an integer of 1 to 1,000, m is an integer of 10 to 1,000, theN-hydroxysuccinimide moiety is a functional group able to chemicallyreact with proteins and peptides including exendins, and Z is (CH₂)_(S)or (CH₂)_(S)NHCO(CH₂)_(S), serving as a linker between exendin andpolyethyleneglycol, wherein s is an integer of 1 to 6.)

In accordance with another aspect thereof, the present inventionpertains to a method for preparing an exendin modified singly with thepolyethyleneglycol or polyethyleneglycol derivative, comprising reactingpolyethyleneglycol or a derivative thereof with exendin in the presenceof a reducing agent in a solvent (step 1); storing the reaction mixtureof step 1 at a predetermined temperature for a predetermined period oftime in the dark (step 2); removing unreacted materials from thereaction mixture (step 3); and separating and purifying an exendinmodified singly with polyethyleneglycol or its derivative from thereaction mixture free of unreacted materials (step 4).

In an embodiment of the present invention, the reaction molar ratio ofpolyethyleneglycol or a derivative thereof to exendin is preferablywithin the range of 1:1-4, depending on the kind of polyethyleneglycolor its derivative. For example, methoxypolyethylene glycolpropionaldehyde is preferably used in an amount of 1 to 2 moles per moleof PEG or its derivative. In the case of methoxypolyethylene glycolsuccinimidyl propionate, its amount preferably ranges from 1 to 4 molesper mole of PEG or its derivative. The solvent used in step 1 may be PBSor an organic solvent. DMSO is preferable. The molar ratio may beselected depending on various factors, including the molecular structureand molecular weight of PEG or its derivative, pH, reaction temperature,and reaction time.

In step 1, a reducing agent is further used. Examples of the reducingagent include NaCNBH₃ or triethylamine, but are not limited thereto.

Further, no particular limitations are imposed on the buffer. It may beany one that is usually used in the art. The buffer may be selected tobe appropriate for the reaction conditions for polyethylene glycol.

Like the reaction molar ratio, it is preferable that the storagetemperature and time are properly controlled according to the kind ofpolyethylene glycol or its derivative. The modified exendin may bestored, for example, at 4° C. for 2 hours or at room temperature for ashorter period of time. This is relevant to the reactivity ofpolyethylene glycol or its derivative. During the storage, modificationoccurs. After a suitable period of time, the reaction can be terminatedwith glycine.

In another embodiment, the unreacted materials of step 3 can be removedthrough a typical method. For example, dialysis in a suitable buffer,such as PBS (phosphate buffered saline), may be used.

Further, the separation and purification of step 4 may be performedusing size extrusion chromatography or reverse high performance liquidchromatography.

In accordance with a further aspect thereof, the present inventionpertains to a pharmaceutical composition, comprising exendin-4, modifiedsingly with PEG or its derivative as an active ingredient, for thetreatment and prevention of diseases caused by the over-secretion ofinsulin, a decrease in plasma glucose level, the inhibition of gastricor intestinal motility, the promotion of satiety, or the inhibition offood intake.

In an embodiment of the present invention, exendin-4 can be increased inhalf life with the retention of natural biological activity throughmodification at a predetermined position, preferably at lysine 27, withpolyethylene glycol or a derivative thereof. In addition, themodification position and the number of PEG or its derivative arerestricted so as to minimize the side effects caused by a variety ofcombinations of such factors.

As a result, the exendin, modified singly with polyethylene glycol or apolyethylene glycol derivative, is useful in the prevention andtreatment of diseases caused by the over-secretion of insulin, such asdiabetes and obesity, and due to a decrease in plasma glucose level, theinhibition of gastric or intestinal motility, the promotion of satiety,or the inhibition of food intake, such as irritable colon syndrome.

The composition comprising PEG-modified exendin as an active ingredientin accordance with the present invention may be formulated into variousoral or parenteral dosage forms.

For the formulation of the active ingredient, a diluent or an expedient,such as a filler, a thickener, a binder, a wetting agent, adisintegrant, a surfactant, etc., may be typically used. Examples oforal solid dosage forms include tablets, pills, powders, granules, andcapsules. These solid forms are usually formulated with at least oneexpedient, such as starch, calcium carbonate, sucrose, lactose, orgelatin. In addition to the expedients, a lubricant, such as magnesiumstearate or talc, may be used. Examples of oral dosage liquid formsinclude suspensions, liquids for internal use, emulsions, and syrups.These dosage forms may include various expedients, such as a wettingagent, a sweetener, a flavor, and/or a preservative, in addition to asimple diluent, such as liquid or liquid paraffin. Examples ofparenteral dosage forms include sterile aqueous solutions, non-aqueoussolvents, suspensions, emulsions, freeze-dried agents, andsuppositories. As for non-aqueous solvents or suspensions, theirexamples include propylene glycol, PEG, vegetable oil, such as oliveoil, and injectable ester such as ethyl oleate.

The dosage amount of the composition according to the present inventionvaries depending on various factors, including body weight, age, sex,body condition, diet, administration time, administration route,excretion rate, and disease severity. Generally, an effective amount maybe completely used through several administrations over one or twoweeks. Within an effective daily dosage, the composition of the presentinvention may be administered once, or in multiple doses per day.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as the limit of the present invention.

MODE FOR THE INVENTION Example 1 Preparation of Methoxypolyethylenepropionaldehyde Coupled Exendin-4

To 0.5 ml of exendin-4 (American Peptide, 1 mg/ml in 50 mM sodiumacetate, pH 5.5) was added methoxypolyethylene propionaldehyde (Nektar,mPEG-pro-pionaldehyde, mPEG-ALD, 2 kDa, 0.95 mg/ml in 50 mM sodiumacetate, pH 5.5), followed by the addition of 20 mM NaCNBH₃ as areducing agent. The molar ratio of mPEG-ALD to exendin-4 was 1:1-2.Reaction between mPEG-ALD and exendin-4 was conducted for 2 hours at 4°C. in the dark. The reaction was terminated with an 0.1% aqueoustrifluoroacetic acid (TFA) to afford an exendin modified at theN-terminus with PEG.

Example 2 Preparation of Methoxypolyethylene Glycol SuccinimidylPropionate-Modified Exendin-4

Exendin-4 (American Peptide, in DMSO) was mixed with an equal volume of2, 5, 10, or 20 kDa methoxypolyethylene glycol succinimidyl propionate(Nektar, mPEG-succinimidyl propionate (mPEG-SPA) in DMSO). Also, anequal volume of 9% tri-ethylamine (TEA in DMSO) was added as a reducingagent in an amount that formed a final concentration of 3%. Theresulting mixture was allowed to react for about 60 min at roomtemperature in the dark. The molar ratio of exendin-4 to mPEG-SPA was1-4:1. The reaction was terminated by adding a 0.1% TFA solution indistilled water at an equal volume to the reaction mixture to produceexendin-4 modified with 2, 5, 10 or 20 kDa PEG-modified exendin-4(respectively, PEG2K-exendin-4, PEG5K-exendin-4, PEG10K-exendin-4 andPEG20K-exendin-4).

Example 3 Preparation of Straight, Bi-Branched or Tri-BranchedPEG-modified Exendin-4

Exendin modified at the N-terminus with PEG was prepared in a mannersimilar to that of Example 2, with the exception that straight (20 kDa),bi-branched (20 kDa) or tri-branched (23 or 40 kDa) methoxypolyethyleneglycol N-hydroxysuccinimide (Nektar) was used.

Experimental Example 1 Separation, Purification and Identification ofPosition Isomer of Exendin-4 Modified Singly with PEG

In order to separate PEG-modified exendin-4, an experiment was carriedout as follows.

The PEG-modified exendin-4 prepared in Examples 1˜3 were separated bycolumn chromatography using a Lichrospher RP-8 column (250×4.0 mm, 5 μm,Merk, Germany) with a mixture of a 0.1% TFA solution in acetonitrile anda 0.1% TFA solution in distilled water serving as a mobile phase. Themobile phase consisted of 35-45% of solvent B (acetonitrile containing0.1% TFA) and 55-65% of solvent A (0.1% distilled water containing 0.1%TFA), and solvent A and solvent B were linearly changed. The exendin-4eluted from the column chromatography was quantitatively analyzed byreading peaks at 215 nm with a UV spectrophotometer. In order to examinethe number of the PEG conjugated to exendin-4, the PEG-modifiedexendin-4 separated through column chromatography was subjected to massanalysis using MALDI-TOF (Matrix assisted laser desorption/ionizationtime-of-flight) mass spectrometry. Each position isomer was digestedwith lysine-C endopeptidase before MALDI-TOF mass spectrometry. Each PEGconjugate was dissolved at a concentration of 1 mg/ml in 50 μl oftriethylamine-HCl buffer (10 mmol/l; pH 7.4) to which 50 μl of theenzyme (1 mg/ml) was added, followed by enzymatic digestion at 37° C.for 1 hour. 5 μl of 10% (v/v) TFA was added to terminate the digestionof lysine-C, and the digests were analyzed using MALDI-TOF massspectrometry. The results are shown in FIGS. 1 to 3 and Table 1.

TABLE 1 Calculated weight Measured in MALDI-TOF Position of PEGExendin-4 PEG Exendin-4 PEG Modification Mass Mass Mass Mass Lys-271278.3 5127.4 1282.5 5120.5 N-terminus 1921.2, 1024.1 3478.3 1988.4,1055.9 3488.4 Lys-12 1024.1 5381.5 1064.4 5399.5

FIG. 1 is an HPLC chromatogram of the exendin modified at the N-terminuswith PEG. As can be seen in the chromatogram, the reaction mixture wasseparated into two different materials which were identified asunreacted exendin-4 and N-terminally PEG modified exendin-4,respectively. In addition, the N-terminally PEG modified exendin-4 wasmeasured to be 98% or higher in purity.

FIG. 2 is an HPLC chromatogram of the products resulting from thereaction of PEG with lysine residues of exendin-4. As seen in thischromatogram, the reaction mixture of Examples 2 and 3 was composed offour different materials. As can be seen in FIG. 3 and Table 1, thesefour materials were analyzed to be unreacted natural exendin-4,exendin-4 modified at lysine-27 with PEG (Lys²⁷-PEG2k-exendin-4),exendin-4 modified at lysine-12 with PEG (Lys¹²-PEG2k-exendin-4), andexendin-4 modified at lysine-12 and 27 with PEG(Lys^(12,27)-PEG2k-exendin-4). The products finally obtained had apurity of 98% or higher.

FIG. 4 shows MALDI-TOF spectra of the exendin-4 modified singly atlysine-27 with PEG in accordance with the present invention. In Example2, PEG-exendin-4 conjugates having various molecular weights wereproduced as various molecular weights of PEG were used.

Experimental Example 2 Biological Activity of PEG-Exendin-4 ConjugatesAccording to Position of PEG Modification

In order to examine the biological activities of natural exendin-4 andPEG-modified exendin-4, experiments were carried out as follows.

Experimental Example 2-1 Assay for Promoting Insulin Secretion

Cells used for this assay were pancreatic islet cells of Sprague Dawleyrats, which could be isolated by collagenase digestion and then Ficollgradient centrifugation. After being cultured for 2˜3 days in anincubator, the isolated pancreatic islet cells were suspended in 1 ml ofKRH buffer (16.7 mM glucose) at a density of 20 cells per well into24-well plates. Afterwards, natural exendin and exendin-4 isomersmodified at the N-terminus, lysine-12 and lysine-27 with PEG(N^(ter)-PEG2k-exendin-4, Lys¹²-PEG2k-exendin-4, andLys²⁷-PEG2k-exendin-4) were added at a concentration of 0.1, 1, 10, and100 nM to each well, respectively, followed by incubation for 2 hours.Then, 2001 of the culture was taken from each well and analyzed forinsulin level using an insulin ELISA kit. The results are shown in FIG.5.

As seen in FIG. 5, the insulin secretory capacity ofLys²⁷-PEG2k-exendin-4 was superior to that of the other modifiedexendin-4, and similar to that of natural exendin-4.N^(ter)-PEG2k-exendin-4 and Lys¹²-PEG2k-exendin-4 were measured to showlow insulin secretory capacity, with N^(ter)-PEG2k-exendin-4 beingpoorer. In addition, natural exendin-4, Lys²⁷-PEG2k-exendin-4,Lys¹²-PEG2k-exendin-4 and N^(ter)-PEG2k-exendin-4 were found to haveEC₅₀ values of 2.94, 3.11, 9.65 and 135.5 nM, respectively.

Experimental Example 2-2 Assay for Receptor Binding

In order to examine the interaction between the ligands exendin-4 andPEG-modified exendin-4 and the GLP-1 receptor, experiments were carriedout as follows.

The insulin secretory cell line INS-1 was plated at a density of 2.5 10⁵cells per well onto 12-well plates and allowed to stably adhere to thebottom of the plates through incubation for about 2 days. After celladhesion, the culture medium was substituted with a binding buffer towhich ¹²⁵I-exendin-4 (an exendin derivative stretching from amino acidresidue 9 to amino acid residue 39) was then added in such an amount asto form a final concentration of 30 μM. Thereafter, natural exendin-4,Lys²⁷-PEG2k-exendin-4, Lys¹²-PEG2k-exendin-4 and N^(ter)-PEG2k-exendin-4were individually added in such an amount as to form a finalconcentration of 0.001˜1000 nM, followed by allowing them tocompetitively bind to the receptor for 2 hours at room temperature.Cells were washed three times with cold PBS to remove unbound¹²⁵I-exendin-4. Finally, cells were disrupted with lysis buffer andmeasured for cell-bound exendin-4 level using a gamma counter. Theresults are shown in FIG. 6.

As seen in FIG. 6, the competitive binding of ¹²⁵I-exendin-4 wasdecreased as the sample increased in concentration. In addition, theposition isomers varied in binding intensity, depending on modificationposition. Natural exendin-4, Lys²⁷-PEG2k-exendin-4,Lys¹²-PEG2k-exendin-4 and N^(ter)-PEG2k-exendin-4 were found to have anIC₅₀ value of 0.1, 1.8, 3.9 and 36.5 nM (concentrations when 50%¹²⁵I-exendin-4 was bound), respectively.

As apparent from these data obtained through cell culture and receptorbinding assay, the PEG-modified exendin-4 isomers were found to besuperior in biological effect and efficiency in the following order:Lys²⁷-PEG2k-exendin-4>Lys¹²-PEG2k-exendin-4>N^(ter)-PEG2k-exendin-4.

Experimental Example 2-3 Animal Model Test

In order to examine the activity of natural exendin-4 and PEG conjugatesin animal models, saline (control), natural exendin-4, and PEG-modifiedexendin-4 (Lys²⁷-PEG2k-exendin-4, Lys¹²-PEG2k-exendin-4 andN^(ter)-PEG2k-exendin-4) were subcutaneously injected in a dose of 1nmol/kg (dosage volume 100 l) into 6-week-old, male db/db mice (modelmice suffering from diabetes)(C57/BLKS/J-db/db, Korea Research Instituteof Bioscience and Biotechnology) 30 min before oral administration with200 mg/ml of glucose (dosage volume 2001). 30 min before, at the timeof, and 15, 30, 60, 120 and 180 min after the oral administration ofglucose, blood samples were taken from the tail vein and monitored forglucose level. The results are shown in FIGS. 7 and 8.

In the control, which was injected only with saline, as seen in FIG. 7,the blood glucose level increased sharply upon glucose administrationand then decreased slowly. In contrast, the groups injected with naturalexendin-4 and PEG-modified exendin-4 underwent a relatively slowincrease in blood glucose level and a relatively rapid decrease in bloodglucose level. Also, there are notable differences in blood glucoselevel between the groups administered with natural exendin-4 andPEG-modified exendin-4, which are believed to result from the fact thatthere are differences in activity and receptor binding ability betweenthe PEG conjugates. Areas under the glucose curve plotted from 0 to 180min on the basis of the result of FIG. 7 are shown in FIG. 8.

As seen in FIG. 8, Lys²⁷-PEG2k-exendin-4 was observed to have activitysimilar to that of natural exendin-4. Lys¹²-PEG2k-exendin-4 andN^(ter)-PEG2k-exendin-4 showed low effects. This difference is believedto be attributed to the fact that there is a difference in biologicalactivity among them.

Accordingly, the data obtained in the biological activity and animalmodel test of Experimental Example 2 demonstrate that the PEG conjugatewith a PEG molecule linked to lysine 27 can decrease blood glucoselevels at higher efficiency than can the other PEG conjugates.

Experimental Example 3 Analysis of Exendin-4 Modified Singly atLysine-27 with PEG for Biological Activity According to Molecular Weightof PEG

In order to examine the biological activity of the exendin-4, which wasmodified at lysine-27 with PEG in a manner similar to that ofExperimental Example 1, according to the molecular weight of PEG, thefollowing experiments were carried out.

Experimental Example 3-1 Promotion of Insulin Secretion

The same procedure as in Experimental Example 1-1 was conducted, withthe exception that the exendin-4 modified with 2, 5, 10 and 20 kDaPEG-modified exendin-4 prepared in Example 2 (PEG2K-exendin-4,PEG5K-exendin-4, PEG10K-exendin-4 and PEG20K-exendin-4) wereadministered at a dose of 10 nM to rat pancreatic islet cells isolatedfrom rats. The results are shown in FIG. 9.

As shown in FIG. 9, the exendin-4 modified with PEG having a molecularweight of 5 kDa or less was not substantially different in biologicalactivity from natural exendin-4. In contrast, the conjugates wereobserved to gradually decrease in biological activity with an increasein the molecular weight of the PEG. When modified with 20 kDa PEG,exendin-4 was measured to show 40% or higher of residual activity.

Experimental Example 3-2 Assay for Receptor Binding

A procedure similar to that of Experimental Example 1-2 was conducted,with the exception that the PEG conjugates, PEG2K-Ex-4, PEG5K-Ex-4,PEG10K-Ex-4 and PEG20K-Ex-4, prepared in Example 2, were administered ata dose of 0.001-1000 nM to the insulin secretory cell line INS-1. Theresults are shown in FIG. 10.

It is apparent from the data of FIG. 10 that the modified exendin-4decreases in receptor binding capacity as the PEG increases in molecularweight. Exendin-4 and PEG-modified exendin-4 were measured to have IC₅₀values of 0.1 nM (natural exendin-4), 1.8 nM (PEG2k-exendin-4), 2.4 nM(PEG5k-exendin-4), 5.6 nM (PEG10k-exendin-4) and 10.7 nM(PEG20k-exendin-4). Hence, it is revealed through this experiment thatthe interaction between the PEG modified exendin-4 and the receptorbecomes weak as the PEG increases in molecular weight, which incurs adecrease in the biological activity of exendin-4, that is, insulinsecretory capacity. When modified at lysine-27, even with 20 kDa PEG(IC₅₀=10.7 nM), the exendin-4 was found to show higher receptor bindingcapacity than was the exendin modified at the N-terminus with lowmolecular weight PEG (IC₅₀=36.5 nM), as is apparent from the data ofFIG. 6, obtained using exendin-4 modified with 2 kDa PEG.

Consequently, when modified at lysine-27, the PEG-modified exendin-4retains the biological activity thereof even though the PEG has a largemolecular weight.

Experimental Example 3-3 Measurement of Anti-Diabetic Activity inAnimals

In order to examine the activity of natural exendin-4 and PEG-modifiedexendin-4 in animal models, saline (control), natural exendin-4, and themodified exendin-4 conjugates PEG2K-Ex-4, PEG5K-Ex-4, PEG10K-Ex-4 andPEG20K-Ex-4 prepared in Example 2 were subcutaneously injected into maledb/db mice (mean blood glucose leve1250 mg/dL) which were 6˜7 weeks old(C57/BLKS/J-db/db, Korea Research Institute of Bioscience andBiotechnology), followed by monitoring levels in the blood taken fromthe tail vein. The results are shown in FIG. 11.

As seen in FIG. 11, when injected into experimental animals afflictedwith diabetes, exendin-4 and PEG-modified exendin-4 acted to induceinsulin secretion in the body to sharply decrease plasma glucose tonormal levels (<150 mg/dL). The control (injected with saline) continuedto have high blood glucose levels. Natural exendin-4 and PEG-modifiedexendin-4 were shown to differ from each other in the retention periodof normal blood glucose level. There is no substantial difference inblood glucose level-decreasing behavior between exendin-4 andPEG-modified exendin. In both the groups administered respectively withexendin-4 and PEG2k-exendin-4, blood glucose was maintained at normallevels until about 6 hours after the injection, and then returned tohigh levels. The exendin-4 modified with PEG5k or PEG10k showed abehavior of decreasing blood glucose levels at higher efficiency thandid natural exenden-4, and was observed to keep blood glucose at normallevels for 12 and 18 hours, respectively. A significantly differentbehavior of decreasing blood glucose levels was detected inPEG20k-exendin-4, in which 20 kDa PEG was coupled. When administered atthe same dosage, PEG20k-exendin-4 was shown to keep the normal bloodglucose level for about 36 hours, which is twice that ofPEG10k-exendin-4 and six times that of natural exendin-4. Thus, theexendin-4, modified at lysine 27 with a high molecular weight PEG, canbe used as a long-acting anti-diabetic drug the medicinal efficacy ofwhich can last for a prolonged period of time.

Experimental Example 3-4 Pharmacokinetic Behavior in Animal

As demonstrated in the above-described experiment, the exendin-4modified at lysine-27 with a high molecular weight PEG showed theproperties of a long-acting peptide drug. In order to examine thelong-acting properties, exendin-4, PEG2k-exendin-4 and PEG20k-exendin-4were observed for pharmacokinetic behavior.

SD rats weighing 200 g were subcutaneously injected with samples at adose of 1 nmol/rat (4.18 μg/rat), followed by sampling blood through atube inserted into the jugular vein. The blood samples were monitoredfor drug level using an ELISA kit. The results are given in FIG. 12.

As is apparent from the data of FIG. 12, natural exendin-4 andPEG2k-exendin-4 showed similar pharmacokinetic behaviors, in which theblood level peaked 30-45 min after administration, then sharplydecreased within a short period of time, and reached a basal line (<2ng/mL) 4 hours after administration. Using this blood concentrationplot, exendin-4 and PEG2k-exendin-4 were calculated to have anelimination half life of 1.6 0.4 and 1.5 0.3 hours, respectively. Incontrast, when PEG20k-exendin-4 was injected, its blood level wasobserved to gradually increase and peak about 12 hours after injection,before slow reduction. The elimination half life of PEG20k-exendin-4 wascalculated to be 22.0±1.7 hours.

Accordingly, the property of long-acting medicinal effect ofPEG20k-exendin-4 is based on the pharmacokinetic behavior thereof,especially the long elimination half life.

Experimental Example 4 Analysis of Exendin-4 Modified Singly atLysine-27 with PEG for Biological Activity According to MolecularStructure of PEG

Through the experiments of Experimental Examples 1-3, the exendin-4modified at lysine-27 with 20 kDa or higher molecular weight PEG wasobserved to exhibit the properties of a long-acting drug. Subsequently,in order to examine the influence of the molecular structure of the PEGon the efficacy and effect of the drug PEG molecules having differentstructures were linked to lysine-27 of exendin-4, and the resultingconjugates were analyzed for biological activity.

Experimental Example 4-1 Assay for Receptor Binding

Receptor binding capacity was measured in the same manner as inExperimental Example 2-1, with the exception that the cells were treatedwith exendin-4 modified with bi-branched PEG (20 kDa) and tri-branchedPEG (23 kDa) prepared according to Example 3. The results are shown inFIG. 13.

As seen in FIG. 13, the binding capacity of PEG-modified exendin-4 wasmeasured to be higher when using tri-branched PEG than when usingbi-branched PEG. The PEG conjugates were calculated to have IC₅₀ valuesof 80.6 nM (tri-branched) and 280.8 nM (bi-branched), respectively.

Experimental Example 4-2 Measurment of Anti-Diabetic Activity in Animal

The same procedure as in Experimental Example 3-3 was repeated, with theexception that the cells were treated with exendin-4 modified withbi-branched PEG and tri-branched PEG prepared according to Example 3.The results are shown in FIG. 14.

As can be seen in FIG. 14, when injected with the bi-branched PEGconjugate, the animals maintained blood glucose at normal levels untilabout 8 hours after the injection, and then returned to high levels.This is believed to be attributable to the fact that the bi-branched PEGconjugate has very low receptor binding capacity (IC₅₀=280 nM, refer toFIG. 13). In contrast, all of the tri-branched PEG conjugates showed aneffect of decreasing blood glucose levels over 48 hours and thus can beused as long-acting drugs. The efficacy of tri-branched PEG conjugatesis based on the pharmacokinetic behavior thereof, especially theelimination rate and distribution thereof.

Therefore, tri-branched PEG modified exendin-4 was observed to have themost effective long-acting drug property.

Experimental Example 4-3 Pharmacokinetic Behavior

The same procedure as in Experimental Example 3-3 was repeated, with theexception that bi-branched PEG conjugates (20 kDa) and tri-branched PEGconjugates (23 kDa) prepared according to Example 3 were intravenouslyinjected. The results are shown in Table 2 and FIG. 15.

TABLE 2 PEG23k-Exendin-4 PEG20k-Exendin-4 (tri-branched) (bi-branched)AUC(ng hr/ml) 7659 ± 1365* 5717 ± 1015* Elimination Half Life(hr) 25.1 ±9.1  28.0 ± 10.4  Volume of Distribution(ml) 9.41 ± 1.57* 25.45 ± 16.84 Volume of distribution(L/kg) 0.05 ± 0.01* 0.13 ± 0.08  Elimination rate(ml/hr) 0.54 ± 0.09* 0.63 ± 0.08* Elimination rate(ml/hr/kg) 2.69 ±0.45* 3.16 ± 0.39*

As can be seen in the data of Table 2, the exendin-4, modified withtri-branched PEG, exhibited lower elimination rates than did theexedin-4 modified by bi-branched PEG. A far lower volume of distributionwas detected in the exendin-4 modified with tri-branched PEG than withbi-branched PEG, indicating that the tri-branched PEG modified exendin-4can exist most stably in the blood. In addition, although there is nosignificant difference in elimination half life between the exendin-4modified with two kinds of PEG, the exendin-4 modified with tri-branchedPEG exhibited the lowest elimination rate and the highest stability.

As can be seen in FIG. 15, the bi- or tri-branched PEG modifiedexendin-4 prepared according to Example 3 gradually decreased in bloodlevel over a long period of time, but at different rates depending onthe structure of PEG.

Experimental Example 4-4 Assay for Promoting Insulin Secretion

The same procedure as in Experimental Example 2-1 was repeated, with theexception that the cells were treated with 0.1, 1, 10 and 100 nM ofnatural exendin-4 or tri-branched PEG modified exendin-4. The resultsare shown in FIG. 16.

As can be seen in FIG. 16, both natural exendin-4 and tri-branched PEGmodified exendin-4 promote insulin secretion in a dose-dependent manner.

Experimental Example 5 Analysis of Exendin-4, Modified Singly atLysine-27, with PEG for Biological Activity According to MolecularWeight of PEG Experimental Example 5-1 Measurment of Anti-DiabeticActivity in Animal

The same procedure as in Experimental Example 3-3 was repeated, with theexception that the cells were treated with 23 or 43 kDa tri-branchedPEG-modified exendin-4 (PEG23k-Ex-4 or PEG43k-Ex-4). The results areshown in FIG. 17.

As can be seen in FIG. 17, the molecular weight difference between 23and 43 kDa PEG did not have any influence on the long-acting property ofthe modified exendin-4. In fact, both of the modified exendin-4sexhibited similar long-acting properties. Accordingly, the datademonstrate that tri-branched PEG having a molecular weight of 20 kDa orhigher guaratees medicinal efficacy over a long period of time.

Experimental Example 5-2 Measurement of Exendin-4 Modified at Lysine-27with Tri-Branched PEG for Anti-Diabetic Activity According to Dose

The same procedure as in Example 3-3 was repeated, with the exeptionthat the cells were treated with 23 kDa tri-branched PEG modifiedexendin-4 (PEG23k-Ex-4) at a dose of 10 nmol/kg or higher. The resultsare shown in FIG. 18.

As can be seen in FIG. 18, the medicinal efficacy of the modifiedexendin-4 was observed to last for about 12-18 hours at a dose of 5nmol/kg for about 36-48 hours at a dose of 10 nmol/kg and for about72-96 hours at a dose of 20 nmol/kg. When administered at a dose of 50nmol/kg the modified exendin-4 showed useful medicinal effects forperiods as long as 120 hours.

Below, a description will be given of formulation examples of thecomposition according to the invention.

Formulation Example Preparation of Pharmaceutical Formulations

Pharmaceutical formulations comprising the exendin-4 modified singlywith PEG were prepared as follows.

1. Preparation of Powder

Exendin-4 singly modified with PEG 2 g

Lactose 1 g

These ingredients were admixed and loaded into an airtight sac toprepare a powder form.

2. Preparation of Tablet

Exendin-4 singly modified with PEG 100 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

These ingredients were admixed and tabletted according to a typicalmethod to produce tablets.

3. Preparation of Capsule

Exendin-4 singly modified with PEG 100 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

These ingredients were admixed and loaded into gelatin capsulesaccording to a typical method to produce capsules.

4. Preparation of Injection

Exendin-4 singly modified with PEG 10 μg/ml

Diluted HCl BP until pH 3.5

NaCl for injection BP up to 1 ml

The exendin-4 singly modified with PEG was dissolved in a suitablevolume of injectable NaCl BP and adjusted to pH 3.5 with diluted HCl BP.The volume was also adjusted with injectable NaCl BP, followed bysufficiently mixing the ingredients. The solution was loaded into 5 mltype I glass ampules which were then melted to seal them.

1. An exendin singly modified with polyethylene glycol or a polyethyleneglycol derivative.
 2. The exendin according to claim 1, wherein theexendin is based on natural or recombinant exendin.
 3. The exendinaccording to claim 1, wherein the exendin is based on exendin-4.
 4. Theexendin according to claim 1, wherein the polyethylene glycol or thepolyethylene glycol derivative is linked to lysine-27 of the exendin. 5.The exendin according to claim 1, wherein the polyethylene glycol or thepolyethylene glycol derivative is a straight type or a branched type. 6.The exendin according to claim 5, wherein the polyethylene glycol is amulti-branched type having two or more branches.
 7. The exendinaccording to claim 6, wherein the polyethylene glycol is a tri-branchedtype.
 8. The exendin according to claim 1, wherein the polyethyleneglycol or the polyethylene glycol derivative ranges in molecular weightfrom 2 to 60 kDa.
 9. The exendin according to claim 8, wherein thepolyethylene glycol or the polyethylene glycol derivative ranges inmolecular weight from 20 to 45 kDa.
 10. The exendin according to claim5, wherein the polyethylene glycol derivative is at least one selectedfrom a group consisting of methoxypolyethylene glycolsuccinimidylpropionate, methoxypolyethylene glycol N-hydroxysuccinimide,methoxypolyethylene glycol propionaldehyde, methoxypolyethylene glycolmaleimide and muti-branched types thereof.
 11. The exendin according toclaim 10, wherein the polyethylene glycol derivative is selected from agroup consisting of methoxypolyethylene glycol succinimidylpropionate,methoxypolyethylene glycol N-hydroxysuccinimide, methoxypolyethyleneglycol propionaldehyde, bi-branched methoxypolyethylene glycolN-hydroxysuccinimide and a tri-branched polyethylene glycol derivativerepresented by the following chemical formula 1:

(wherein n is an integer of 1 to 1,000, m is an integer of 10 to 1,000,the N-hydroxysuccinimide moiety is a functional group able to chemicallyreact with proteins and peptides including exendins, and Z is (CH₂)_(S)or (CH₂)_(S)NHCO(CH₂)_(S), serving as a linker between exendin andpolyethyleneglycol, wherein s is an integer of 1 to 6.)
 12. A method forpreparing an exendin modified singly with the polyethyleneglycol orpolyethyleneglycol derivative, comprising: reacting polyethyleneglycolor a derivative thereof with exendin in the presence of a reducing agentin a solvent (step 1); storing the reaction mixture of step 1 at apredetermined temperature for a predetermined period of time in the dark(step 2); removing unreacted materials from the reaction mixture (step3); and separating and purifying an exendin modified singly withpolyethyleneglycol or its derivative from the reaction mixture free ofunreacted materials (step 4).
 13. The method according to claim 12,wherein the solvent used in step 1 is phosphate buffer saline or anorganic solvent.
 14. The method according to claim 13, wherein theorganic solvent is DMSO.
 15. The method according to 12, wherein thereaction molar ratio of the polyethyleneglycol or polyethylene glycolderivative thereof to exendin is preferably within the range of 1:1˜4.16. A pharmaceutical agent for the prevention and treatment of a diseasecaused by the over-secretion of insulin, comprising as an activeingredient the exendin singly modified with polyethylene glycol or apolyethylene glycol derivative of claim
 1. 17. A pharmaceutical agentfor the prevention and treatment of a disease caused by a decrease inplasma glucose level, the inhibition of gastric or intestinal motility,the promotion of satiety, or the inhibition of food intake, comprisingas an active ingredient the exendin singly modified with polyethyleneglycol or a polyethylene glycol derivative of claim
 1. 18. Thepharmaceutical agent according to claim 16, wherein the disease isdiabetes or obesity.
 19. The pharmaceutical agent according to claim 17,wherein the disease is irritable colon syndrome.